Early diagnosis is a priority and highly desirable objective in all fields of medicament, particularly because it allows an appreciable improvement in the patient's life and a concomitant saving on the part of health care systems or on the part of the actual patients. In the particular case of the invention described herein, early diagnosis is that of potential or existing Toxoplasma gondii infection in pregnant women, with particular concern for the health of the foetus, and in infected subjects, particularly those with impaired immunity.
Toxoplasma gondii is an obligate intracellular parasite that infects all mammalian cells, including those of human subjects (McCabe and Remington, N. Engl. J. Med. 1988, 318–313–5), and other animal genera, e.g. birds. The life cycle of the parasite is complex and one may distinguish between three stages of infection: tachyzoite (asexual), bradyzoite (in tissue cysts, asexual) and sporozoite (in oocysts, sexual reproduction). Transmission typically occurs through ingestion of undercooked meat harbouring tissue cysts or vegetables contaminated with oocysts shed by cats. Human infection is generally asymptomatic and self-limiting in immunocompetent hosts. In contrast, in subjects with impaired immunity (particularly those affected by AIDS), toxoplasmosis is a severe opportunist infection, which may give rise to encephalitis with very serious outcomes (Luft, B. J., Remington J. S., 1992, Clin. Infect. Dis. 15, 211–22). Moreover, contracting primary infection during pregnancy may lead to miscarriages or to severe foetal disease in mammals.
For an extensive overview of the problem of toxoplasmosis the reader is referred to the specialistic medical literature.
Diagnosis of T. gondii infection is established by isolating the micro-organism in the blood or body fluids, identifying the parasite in tissues, detecting specific nucleotide sequences with PCR, or by detecting specific anti-T. gondii immunoglobulins produced by the host in response to the infection (Beaman et al., 1995 Principles and Practice of Infectious Diseases 4th Ed., Churchill Livingstone Inc., New York, 2455–75; Remington J S et al. 1995, Infectious Diseases of the Fetus and Newborn Infant, W. B. Saunders, Philadelphia, Pa., 140–267).
One of the main problems in diagnosing T. gondii infections has to do with pregnant women. To implement suitable therapies in good time and avoid possible damage to the foetus it is very important to establish if parasitic infection occurred before or after conception. This is generally done by attempting to detect the presence of the various classes of anti-Toxoplasma immunoglobulins (IgG, IgM, IgA, avidity of IgG). For this reason, the availability of specific, sensitive diagnostic agents is desirable.
T. gondii antigens have long been known and available, first of all as antigen mixtures obtained in various ways (FR 2,226,468, Mérieux; SU 533376, Veterinary Research Institute; JP 54044016, Nihon Toketsu Kanso), then as subsequent isolations of pure antigens (EP 0 082 745, Mérieux; EP 0 301 961, INSERM, Pasteur; WO 89/5658, Transgene) and their characterisation both as proteins, and of their respective genes (WO 89/08700, U. Leland, Dartmouth Coll.; U.S. Pat. No. 4,877,726, Res. Inst. Palo Alto; WO 89/12683, INSERM, Pasteur; EP 0 391 319, Mochida Pharm.; IT 1,196,817, CNR; EP 0 431 541, Behringwerke; WO 92/01067, CNRS; WO 92/02624, U. Flinders; WO 92/11366, Innogenetics, Smithkline Beecham; U.S. Pat. No. 5,215,917, Res. Inst. Palo Alto; WO 92/25689, FR 2702491, INSERM, Pasteur; WO 96/02654, bioMeriéux, Transgene; EP 0 710 724 Akzo; EP 0 724 016, bioMeriéux; EP 0 751 147, Behringwerke; U.S. Pat. No. 5,633,139, Res. Inst. Palo Alto; WO 97/27300, Innogenetics; U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto; WO 99/32633, Heska; JP 11225783, Yano; WO 99/61906, Abbott; WO 99/66043, Smithkline Beecham; JP 2000300278, Yano; WO 00/164,243, Virsol).
Numerous studies have found various different antigenic proteins of T. gondii and the gene sequences of these have also been determined.
Among the most interesting proteins both for diagnostic and therapeutic purposes, in the form of vaccines, we should mention: the surface antigens SAG1 (or P30) (WO 89/08700, Stanford University; WO 89/12683 Pasteur, INSERM; WO 94/17813, WO 96/02654, Transgene, bioMeriéux; EP 0 724 016, WO 99/61906, U.S. Pat. No. 5,962,654, Harning et al., Clinical and Diagnostic Laboratory Immunology, May 1996, 355–357); SAG2 (or P22) (Parmley et al., 1992, J. Clin. Microbiol. 30, 1127–33); the dense granule proteins GRA1 (or P24) (EP 0 301 961, Pasteur, INSERM; WO 89/05658, Transgene, Cesbron-Delauw, et al., 1989 P.N.A.S. USA 86, 7537–41); GRA2 (or P28) (WO 93/25689, INSERM, Pasteur; U.S. Pat. No. 5,633,139, U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto; Prince et al., Mol. Biochem. Parasitol., 34 3–14); GRA4 (Mevelec et al., Mol. Biochem. Parasitol. 56, 227–38); GRA6 (or P32) (FR 2,702,491, INSERM, Pasteur; Lecordier al., Mol. Biochem. Parasitol. 70, 85–94); GRA7 (WO 99/61906, Abbott; Jacobs et al., Mol. Biochem. Parasitol. 91, 237–49); GRA3 (Robben et al. 2002, J. Biol. Chem. 277, 17544–47): the rhoptry antigens ROP1 (or P66) (U.S. Pat. No. 5,976,553, U. Leland; EP 0 431 541, Innogenetics); ROP2 (or P54) (Sharma et al., J. Immunol., 131, 377–83).
As described in the above-mentioned references, the antigens were obtained with well-known recombinant cDNA techniques in expression vectors. For example, for the antigen SAG1, WO 98/08700 uses known expression vectors in phage λgt11. WO 98/12683 uses the same phage and transfects E. coli with a proprietary plasmid, or by preparing a special expression cassette, as in WO 96/02654. EP 0 724 016 obtains mimotypes, using combinatorial expression libraries of peptides. EP 0 301 961 describes how to obtain excretion-secretion antigens with molecular weights ranging from 20 kDa to 185 kDa. WO 89/05658 describes a protein containing the epitopes of the 24 kDa protein recognised by the antibodies produced against Toxoplasma excretion-secretion antigens; this protein is obtained by transfection of cells by means of expression vectors. The antigen P28 (GRA2) is described in U.S. Pat. No. 5,633,139 and the method of obtaining it is again implemented through expression in phage λgt11. The antigen P32 (GRA6) is described in patent FR 2,702,491, the antigen ROP1 (P66) in U.S. Pat. No. 5,976,553, P35 (or GRA8) in EP 0 431 541, WO 99/57295 and WO 99/61906, and lastly P68 in EP 0 431 541.
It should be stressed that all these antigens are obtained by means of molecular biology techniques that use the expression of proteins in bacterial cells. None of the documents cited describe the technique of expression/exposure of libraries of cDNA deriving from Toxoplasma gondii in the lambda phage (phage display) to obtain fragments of antigens of the pathogen.
The invention described herein uses a new vector of DNA expression and protein exposure as molecular fusion with the amino-terminal part of protein D of the lambda bacteriophage capsid (pD) (λKM4).
The expression/exposure vector was described for the first time in patent application PCT/IT01/00405, filed on 26 Jul. 2001, the most important part of which is incorporated herein. This vector, called λKM4, differs from that used in expression only experiments (λgt11) in that the recombinant protein coded for by the DNA fragment is expressed as fusion with a protein of the bacteriophage itself and then exposed on the capsid. According to the vector project, the phage exposes the protein fragment on the surface only if its open reading frame (ORF) coincides with pD. The size of the fragments of DNA cloned in our libraries was selected in order to represent a population of medium size ranging from 300 to 1000 nucleotide base pairs (bp), and, for statistical reasons, most of the out-of-frame sequences contain stop codons which do not permit their translation and consequently exposure on the surface of the phage.